CN110004125A - A kind of marine bacteria source development of new type alkali-resistant fibre organic solvent-resistant esterase and application - Google Patents

Abstract

The present invention relates to a kind of marine bacteria source development of new type alkali-resistant fibre and organic solvent carboxy-lesterase Eij5 and its encoding gene and applications.Carboxy-lesterase Eij5 encoding gene comes from Erythrobacter jejuensis KCTC23090^TFor involved esterase gene after heterogenous expression, substrate shows highest catalytic activity when being p-nitrophenol butyrate (C4), and catalyzing hydrolysis pH value range is 6.5-10.5, catalyzing hydrolysis temperature range is 25-55 DEG C, is resistant to a variety of organic solvents and metal ion.The esterase has the characteristic of saline-alkali tolerant organic solvent-resistant, can be applied to food processing, chiral drug synthesis and containing it is saline and alkaline, containing the environment remediation and industrial production under the conditions of organic solvent.

Description

A kind of marine bacteria source development of new type alkali-resistant fibre organic solvent-resistant esterase and application

Technical field

The invention belongs to genetic engineering fields, and in particular to a kind of marine bacteria source development of new type alkali-resistant fibre and organic solvent ester Enzyme, its encoding gene and its application.

Background technique

Esterase (EC 3.1.1.2) be it is a kind of can be catalyzed the strong hydrolase for being formed and being broken of ester, be widely present in animal, In plant and microorganism.Microorganism esterase participates in esterification, a variety of biochemical reactions such as transesterification and hydrolysis.Esterase is catalyzed anti- Should have many advantages, such as that reaction condition is mild, reaction efficiency is high, by-product is few, does not need coenzyme, and hydrolyze with extremely strong region Specificity and stereospecificity, therefore, esterase be widely used in food processing, fine chemistry industry, biomedicine, chiral drug and The every field such as environmental improvement.

Deep-sea is a natural microbial resources treasure-house, and deep-sea source esterase usually has the Optimality with environmental correclation Matter, such as thermostabilization, salt tolerant, alkaline-resisting, low temperature resistant etc. are screened esterase with unique properties from marine microorganism and are had become out Send out an important directions of infant industry enzyme.In recent years, with the development of sequencing technologies, many newfound bacterial groups are obtained With by genome sequencing, by bioinformatic analysis to genome annotation after, sequence screening is carried out simultaneously to the albumen of annotation Binding function screening, can filter out some novel enzyme genes with unique physicochemical properties.

The present invention screens a kind of development of new type alkali-resistant fibre and organic solvent esterase gene from a kind of deep-sea bacterium, and to the gene It is recombinantly expressed, recombinase has the characteristics that saline-alkali tolerant, resistance to metal ion, organic solvent-resistant and detergent, can be applied to Food processing, chiral drug synthesis and containing it is saline and alkaline, containing the environment remediation and industrial production under the conditions of organic solvent.

Summary of the invention

The object of the present invention is to provide a kind of new marine bacteria source hydrolase, its encoding gene and preparation method thereof, The hydrolase can be used for the biocatalysis and conversion of esters degradation and other ester type compounds under the conditions of high temperature and high salt.

The present invention relates to a kind of isolated polypeptide with hydrolytic enzyme activities, is selected from the group:

(a) polypeptide, it is consistent with sequence shown in the polypeptide of SEQ ID NO:2；Or

(b) polypeptide, be SEQ ID NO:2 shown in polypeptide separate catalytic center position carry out it is various replace, add and/ Or the mutant that one or several amino acid of missing obtain, the mutant have with protein sequence shown in SEQ ID NO:2 extremely Few 90% or more homology and at least 90% or more hydrolytic enzyme activities.

Polypeptide of the present invention with hydrolytic enzyme activities can express saline-alkali tolerant and organic solvent ester from a kind of The bacterial strain Erythrobacter jejuensis KCTC23090 of enzyme^T, which is isolated from South Korea's Jizhou Island beach seawater, point Class is named as Erythrobacter jejuensis, buys from Taejon, Korea KCTC strain library.

It is screened based on complete genome sequencing from Erythrobacter jejuensis KCTC23090 and obtains hydrolase Gene Eij5, nucleotide sequence is as shown in SEQ ID No.1.Gene Eij5 size is 951bp, and base composition is 165 A (17.35%), 175 T (18.40%), 321 C (33.75%) and 290 G (30.49%), coding albumen size are 316 ammonia Base acid residue, amino acid sequence is as shown in SEQ ID No.2.By hydrolase Eij5 amino acid sequence in GenBank data Homology search is carried out in library, it is not belong to bacterium Erythrobacter litoralis that consistency is highest therewith HTCC2594^TIn esterase, consistency is 83% (its number of registration in GenBank database is WP_011414343.1). Amino acid sequence analysis shows that active site serine is located at the guarantor with glycine-X- serine-X- glycine feature (amino acid position 161-166) is kept in sequence, and with 255 aspartic acids, 285 hyte propylhomoserins are collectively formed in the catalysis of esterase The heart, it is Section IV family esterase conserved features sequence that 89-92 amino acids, which constitute histidine-glycine-Gly-Gly sequence, Column, in conclusion Eij5 is newcomer in ester-type hydrolysis enzyme Section IV family.

It, can be to separate catalytic center amino acid position shown in SEQ ID NO:2 under the premise of not influencing Eij5 protein active It sets the amino acid sequence (preferably away from the amino acid position of 161-166,255 and 285) and carries out various substitutions, additions and/or deletions One or several amino acid, which obtain, has the active derived protein of Eij5.According to the common knowledge of art technology, protein Biological activity be closely related with its functional domain.In general, the position in protein catalyst structure domain occurs Point mutation, may be because having an impact protein 2 and 3 dimensional organization, and then influences its biological activity.And for occurring Amino acid sites far from functional domain (amino acid position of preferably 161-166,255 and 285), since this region is not involved in Individual point mutation of protein function conformation, amino acid will not generate substantial effect to the biological activity of protein, so as to Enough basic biological functions for retaining crude protein.Preferred hydrolase Eij5 mutant have at least with SEQ ID NO:2 institute The homology of 90% or more the amino acid sequence shown more preferably has at least 95% or more homology, most preferably has at least 99% or more homology.The mutant can retain the biological function of hydrolase Eij5, the preferably mutant substantially Enzymatic activity with the hydrolase at least 90% or more with amino acid sequence shown in SEQ ID NO:2 more preferably has at least 95% or more enzymatic activity, most preferably at least 99% or more enzymatic activity.

The invention further relates to the mature polypeptide of SEQ ID NO:2 or its homologous sequences comprising replacing, missing and/or insertion The artificial variants of one or more amino acid, mutated site are preferably smaller than 5, more preferably less than 3, most preferably only 1 position Set the mutation of amino acid.The example of conservative substitution is with the following group: basic amino acid group (arginine, lysine and group ammonia Acid), acidic amino acid group (glutamic acid and aspartic acid), polar amino acid group (glutamine and asparagine), hydrophobic amino Sour group (leucine, isoleucine and valine), aromatic amino acid group (phenylalanine, tryptophan and tyrosine) and p1 amino acid Group (glycine, alanine, serine, threonine and methionine).Usually not changing the amino acid substitution of specific activity is ability Known to domain, and by such as Η .Neurath and R.L.Hill, 1979 in The Proteins, Academic Press, New It is described in York.The exchange most generally occurred be Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn、Ala/Val、Ser/Gly、Tyr/Phe、Ala/Pro、Lys/Arg、Asp/Asn、Leu/Ile、Leu/Val、Ala/ Glu and Asp/Gly etc..

Known mutagenesis, recombination and/or Shuffling Method can be used, then carry out relevant screening process, such as by Reidhaar-Olson and Sauer, 1988, Science, 241:53-57；Bowie and Sauer, 1989, Proc.Natl.Acad.Sci.USA 86:2152-2156；Those of disclosed in WO95/17413 or WO 95/22625, into Row one or more amino acid substitution, missing and/or insertion are simultaneously tested.Other workable methods include fallibility PCR, bite Phage display (such as Lowman etc., 1991, Biochemistry 30:10832-10837；U.S. Patent number 5,223,409； WO92/06204) and regiondirected mutagenesis (region-directed mutagenesis) (Derbyshire etc., 1986, Gene 46:145 and 1988, DNA 7:127).

The invention further relates to isolated polynucleotides, and it includes the hydrolase Eij5 that the coding present invention has hydrolytic enzyme activities Nucleotide sequence, or by coding the present invention have the active mutant of hydrolase Eij5 nucleotide sequence form.

The present invention relates to codings to have the active isolated polynucleotides of hydrolase Eij5, is selected from the group:

(a) polynucleotides, it is consistent with sequence shown in the nucleotide of SEQ ID NO:1；Or

(b) polynucleotides, for in nucleotide sequence shown in SEQ ID NO.1 remove 481-498,763-765 and Other nucleotide outside 853-855 nucleotide are replaced, add and/or lack the mutation that one or several nucleotide obtain Body gene, the polynucleotides have the homology with nucleotide sequence at least 90% or more shown in SEQ ID NO:1.

The invention further relates to isolated polynucleotides, and it includes the nucleotide sequences for encoding hydrolase Eij5 of the present invention.It should Sequence is consistent with nucleotide sequence shown in SEQ ID NO.1；By hydrolase Eij5 amino acid sequence in GenBank nr number According to Homology search is carried out in library, it is not belong to bacterium Erythrobacter litoralis that consistency is highest therewith HTCC2594^TIn esterase, consistency is 83% (its number of registration in GenBank database is WP_011414343.1). The gene coding catalytic active center amino acid codon be located at genes of SEQ ID NO:1 481-498,763-765 and 853-855 base-pair.

The present invention also provides in nucleotide sequence shown in SEQ ID NO.1 remove 481-498,763-765 and 853-855 Other nucleotide outside the nucleotide of position are replaced, add and/or lack one or several nucleotide can base to obtain coding The mutant gene of this reservation hydrolase Eij5 biological activity of albumen.Preferred hydrolase Eij5 mutant gene has at least With the homology of 90% or more nucleotide sequence shown in SEQ ID NO:1, more preferably there is at least 95% or more homology, Most preferably there is at least 99% or more homology.

The invention further relates to the nucleic acid constructs comprising isolated polynucleotides of the invention, can use multi-mode operation perhaps The isolated polynucleotides of hydrolase of the present invention are encoded to provide the expression of hydrolase.The isolated polynucleotides and one or Multiple regulating and controlling sequences are operably connected, and the regulating and controlling sequence is in a suitable host cell in the item compatible with the regulating and controlling sequence The expression of coded sequence is instructed under part.Regulating and controlling sequence can be promoter sequence appropriate, be by for expressing code book hair The nucleotide sequence of the host cell identification of the polynucleotides of bright polypeptide.Promoter sequence contains the transcription of the expression of direct polypeptide Regulating and controlling sequence.Promoter can be any nucleotide sequence that transcriptional activity is shown in selected host cell, including mutation , truncated and heterozygosis promoter, and can be from coding and the homologous or heterologous extracellular or intracellular polypeptide of host cell Gene obtains.Using gene clone technology, the hydrolase Eij5 gene being cloned into can be connected on suitable carrier, and converted Or it is transfected into prokaryotes or eucaryote host expresses preparation and reorganization hydrolase Eij5.Suitable prokaryotes host includes each Kind bacterium such as E.coli etc., suitable eucaryote host include yeast (such as methanol yeast) and mammalian cell (such as China Hamster ovary cell) etc., it is preferred to use prokaryotic expression system E.coli.

The protokaryon or eukaryotic expression for the various commercially viable purchases that suitable carrier is well known to those skilled in the art carry Body, prokaryotic expression carrier such as pET serial carrier, pQE serial carrier；Yeast expression carrier pPICZ- α-A, pHIL-D2, pPIC9, pHIL-S1(Invitrogen Corp.San Diego.California.USA)；Animal cell expression vectors pSVK3, pMSG (Amersham Pharmacia Biotech Inc.USA) etc..

The present invention relates to the nucleic acid constructs comprising isolated polynucleotides of the invention, can be compiled with multi-mode operation is permitted The isolated polynucleotides of code book invention hydrolase are to provide the expression of hydrolase.The isolated polynucleotides and one or more A regulating and controlling sequence is operably connected, and the regulating and controlling sequence is in a suitable host cell in the condition compatible with the regulating and controlling sequence The lower expression for instructing coded sequence.Regulating and controlling sequence can be promoter sequence appropriate, be by encoding the present invention for expressing The nucleotide sequence of the host cell identification of the polynucleotides of polypeptide.Promoter sequence contains the transcription tune of the expression of direct polypeptide Control sequence.Promoter can be any nucleotide sequence that transcriptional activity is shown in selected host cell, including mutation, Truncated and heterozygosis promoter, and can be from the gene of coding and the homologous or heterologous extracellular or intracellular polypeptide of host cell It obtains.

The invention further relates to recombinant host cells, and it includes isolated polynucleotides of the invention, it may be advantageous to be used for water In the recombinant production for solving enzyme Eij5.By the vector introduction host cell comprising polynucleotides of the invention, the selection of host cell It is largely dependent upon gene and its source of coding polypeptide.Host cell can be hydrolase Eij5's of the invention Useful any cell in recombination generation, for example, protokaryon or eukaryotic.Using gene clone technology, the water that can will be cloned into Solution enzyme Eij5 gene is connected on suitable carrier, and converts or be transfected into prokaryotes or eucaryote host expresses preparation weight Group hydrolase Eij5.Suitable prokaryotes host includes various bacteriums such as E.coli etc., can pass through following protoplast transformation Or or electroporation carrier is transformed into prokaryotic cell.Suitable eucaryote host includes yeast (such as methanol yeast) and feeds Newborn zooblast (such as Chinese hamster ovary cell).Present invention preferably employs the hydrolysis of prokaryotic expression system E.coli Expression product Enzyme Eij5.One preferred example is that the hydrolase gene eij5 for screening the present invention is connected to coli expression carrier It on pSMT3, and is transformed into E. coli Rosetta (DE3), the recombinase of high activity is gone out through inducing expression.

The invention further relates to the methods for generating hydrolase Eij5 of the present invention comprising:

(a) recombinant host cell is cultivated under conditions of helping to create hydrolase Eij5, wherein the host cell packet Nucleotide shown in the NO:1 of ID containing SEQ or the nucleotide in its at least one mutational site；

(b) polypeptide is recycled.

In production method of the invention, it is being suitable for generating the hydrolase Eij5's using methods known in the art Cell is cultivated in nutrient medium.For example, can be by suitable culture medium and allowing to express and/or separate the hydrolase In the shaking flask culture carried out under conditions of Eij5 and laboratory or industrial fermentation tank small-scale or large scale fermentation (including even It is continuous, in batches, fed-batch or solid state fermentation) cultivate cell.Using methods known in the art in suitable nutrient medium In cultivated, the nutrient medium includes carbon source and nitrogen source and inorganic salts.Suitable culture medium can be from commercial supplier It obtains or can be prepared according to disclosed composition.If polypeptide is secreted into nutrient medium, which can be from the culture It is directly recycled in base.If polypeptide is not secreted, can be recycled from cell lysate.

Methods known in the art recycling can be used in gained hydrolase Eij5.For example, can be by conventional method from battalion It supports and is recycled in culture medium, the conventional method includes but is not limited to centrifugation, filtering, extraction, spray drying, evaporation or precipitates.It can To be purified by a variety of methods known in the art, the method includes but be not limited to chromatography (for example, ion exchange, affine, thin The methods of water, chromatofocusing and size exclusion) or differential solubility (such as ammonium sulfate precipitation).

The present invention also provides hydrolase Eij5 or the application of the host strain of hydrolase Eij5 industrially can be expressed, such as It can be used for being catalyzed ester-type hydrolysis.Show that hydrolase Eij5 has esterase active by esterase activity measurement.Eij5 or above-mentioned can table Host strain up to Eij5 can be used for hydrolyzing short-chain aliphatic ester, such as the preferred short chain fatty acids of C4-C8 short carbon chain aliphatic ester Rouge is the p-nitrophenyl phenolic ester with C4-C8 short carbon chain, such as p-nitrophenol butyrate, p-nitrophenol capronate and to nitre Base phenol caprylate etc., catalytic activity highest when wherein substrate is p-nitrophenol butyrate (C4), enzyme activity 42.5U/mg.

Eij5 catalyzing hydrolysis temperature range is 25~55 DEG C, preferably 45 DEG C；The pH value of the hydrolysis is 6.5~10.5, Preferably 7.0.Still there is the enzyme activity under the conditions of 0.8mol/L NaCl；In Ba²⁺、Ca²⁺、Mg²⁺And Sr²⁺In the presence of can retain 51%-90% or more activity；Under the conditions of adding organic solvent DMF, DMSO, glycerol, methanol, enzyme activity is basically unchanged, and is being added Under the conditions of EDTA and detergent TritonX-100, the enzyme activity increases.

Bacterium Erythrobacter jejuensis of the present invention from South Korea's Jizhou Island beach sea water origin KCTC23090^TMiddle screening obtains new saline-alkali tolerant organic solvent-resistant hydrolase gene, it was found that the gene coded protein has excellent Good enzymatic property can be applied in catalysis solution ester and enzymatic clarification ester process of producing product.The hydrolase gene of acquisition can gram It is grand that heterogenous expression is realized into suitable host, it realizes industrialized production saline-alkali tolerant organic solvent-resistant hydrolase, is subsequent work Industry application provides low-cost saline-alkali tolerant organic solvent and hydrolyzes enzyme starting material.The production of the enzyme can be at detergent, waste water Important economy and society are shown in the saliferous such as reason, fine chemistry industry, pharmacy and environment remediation and production technology containing organic solvent Value.

Detailed description of the invention

Fig. 1 is the policapram gel electrophoresis analysis figure for purifying hydrolase Eij5.

Fig. 2 is the substrate specificity figure of hydrolase Eij5.C2: p-nitrophenol acetic acid esters；C4: p-nitrophenol butyric acid Ester, C6: p-nitrophenol capronate；C8: p-nitrophenol caprylate；C10: p-nitrophenol decylate；C12: p-nitrophenyl Phenol dodecanoate；C14: p-nitrophenol myristinate；C16: p-nitrophenol Palmitate；Define measured value when substrate is C4 It is 100%.

Fig. 3 is hydrolase Eij5 optimal reaction pH figure.

Fig. 4 is hydrolase Eij5 optimal reactive temperature figure.

Fig. 5 is bivalent cation to hydrolase Eij5 activity influence figure.

Fig. 6 is organic solvent to hydrolase Eij5 activity influence figure.

Specific embodiment

The acquisition of 1 hydrolase gene eij5 of embodiment

Based on the bacterium Erythrobacterjejuensis for being isolated from South Korea's Jizhou Island beach sea water origin KCTC23090^TFull-length genome, open reading frame prediction and gene annotation are as a result, screening lipid hydrolyzing enzyme related gene.Pass through Known hydrolase gene sequence in Blastp (http://blast.ncbi.nlm.nih.gov/) aligned sequences and database Homology.Analysis is compared through database and obtains 951bp, and base composition is 165 A (17.35%), 175 T (18.40%), 321 C (33.75%) and 290 G (30.49%), nucleotide sequence is as shown in SEQ ID No:1.Encoding albumen size is 316 Amino acid residue, amino acid sequence are (its three letter amino acid sequence is as shown in SEQ ID No.2) as follows:

The Eij5 protein sequence is subjected to Homology search in GenBank, Amino acid sequence identity is highest therewith is Bacterium Erythrobacter litoralis HTCC2594 is not belonged to^TIn esterase consistency be that 83% (it is in GenBank number It is WP_011414343.1 according to the number of registration in library).

Phylogenetic Analysis shows that hydrolase Eij5 belongs to esterase Section IV family.Amino acid sequence analysis is shown, living Property site serine be located at glycine-X- serine-X- glycine feature conserved sequence in (amino acid position 161- 166), with 255 aspartic acids, 285 hyte propylhomoserins collectively form the catalytic center of esterase, 89-92 amino acids composition group ammonia Acid-Gly-Gly-Gly conserved sequence.Its amino acid sequence feature meets Section IV family ester-type hydrolysis enzyme feature.

In conclusion Eij5 should be a newcomer of esterase family.

The building of the recombinant expression plasmid and recombinant bacterial strain of 2 gene Eij5 of embodiment

The gene Eij5 that the present invention obtains is cloned on expression vector, recombinant strains are constructed.Based on NCBI ORF The gene open reading frame sequence that the ORF analysis of Finder obtains, upstream primer Eij5F (the 5 '-TCG of design amplification full genome CGGATCCATGAATGACGGCAATCCCTTCG-3 ', BamHI) and downstream primer Eij5R (5 '- TCCCGAGCTCTCAGGCGTTCCCCGCCAGC-3 ', SacI), PCR amplification confirms full length gene sequence.Using enzyme cutting clone Method constructs expression plasmid, that is, uses BamHI and SacI double digestion PCR product, segment after purification with through the bis- enzymes of BamHI and SacI The plasmid pSMT3 connection cut, using CaCl₂Conversion method is converted into E.coli DH5 α, and positive gram of kanamycin resistance screening It is grand.Using the plasmid of plasmid extraction kit (Omega, the U.S.) extraction positive colony, identified through BamHI and SacI double digestion, The DNA fragmentation close to 1000bp or so is obtained, is accredited as gene eij5 through sequencing.Recombinant expression plasmid is transformed into E.coli Rosetta (DE3) is expressed in bacterial strain, building expression recombinant bacterial strain.

Embodiment 3 expresses recombination Eij5 using recombinant strains

The 3ml recombinant strains built are transferred to 100ml contains 50 μ g/ml kanamycins and 34 μ g/ml chlorine are mould In the LB liquid medium of element, 37 DEG C of shaken cultivations to OD₆₀₀Reach 0.6, the IPTG that final concentration of 0.5mM is added is induced Expression, is transferred to 16 DEG C with 150r/min shaken cultivation 20h.Low-temperature centrifugation collects thallus, is resuspended in NTA-10 solution (500mM chlorine Change sodium, 10mM imidazoles, 20mM Tris hydrochloric acid, pH 8.0) in, ultrasonic disruption processing is carried out on ice.On low-temperature centrifugation is collected Clearly, using NTA-Ni²⁺Affinity column chromatography purifying expression albumen.Expressed recombinant protein contains the 6 × His tag of N-terminal, affable It is inhaled on column with layer is adsorbed onto, by the imidazole solution gradient elution of various concentration, collects eluent.Through SDS-PAGE testing goal Distribution situation of the albumen in eluent.The ubiquitin-like SUMO at recombinant protein N end is cut off in bag filter using ULP1 enzyme, and is adopted Use NTA-Ni²⁺Affinity column chromatography removes SUMO albumen, collects sample and carries out SDS-PAGE detection.Obtain electrophoretically pure recombinant protein Eij5, molecular weight about 33kDa (Fig. 1).Protein concentration is measured with Brandford method.

The Activity determination of 4 recombination Eij5 of embodiment

Utilize the recombination hydrolase Eij5 activity of p-nitrophenol butyric acid ester process measurement purifying.Concrete operations: 1ml reactant It include 1mM p-nitrophenol butyrate in system, 100mM potassium dihydrogen phosphate-sodium hydrate buffer solution (pH 7.0) and 4.04ng are pure Zymoprotein, using ultra-violet and visible spectrophotometer (Beckman DU800 type, the U.S.) the METHOD FOR CONTINUOUS DETERMINATION extinction under the conditions of 45 DEG C Value A₄₀₅2min uses the enzyme solution of inactivation as control for returning to zero.One enzyme activity unit is defined as per minute to nitro Phenol ester catalysis generates the required enzyme amount of l μm of ol p-nitrophenol.The esterase active measured is 42.5U/mg.

The analysis of 5 hydrolase Eij5 substrate specificity of embodiment

The substrate specificity analysis of hydrolase Eij5 uses system (1ml): 100mM potassium dihydrogen phosphate-sodium hydroxide buffer 4.04ng pure enzyme protein, the METHOD FOR CONTINUOUS DETERMINATION light absorption value A at 25 DEG C is added in liquid (pH 7.5), 1mM substrate₄₀₅2min.Measurement uses Substrate are as follows: p-nitrophenol acetic acid esters (C2), p-nitrophenol butyrate (C4), p-nitrophenol capronate (C6), to nitre Base phenol caprylate (C8), p-nitrophenol decylate (C10), p-nitrophenol dodecanoate (C12), p-nitrophenol 14 Acid esters (C14), p-nitrophenol Palmitate (C16).Show the Eij5 p-nitrophenyl phenolic ester shorter to acyl group carbochain after measured (C4, C6 and C8) has higher catalytic activity, catalytic activity highest when wherein substrate is p-nitrophenol butyrate (C4).As a result Show that hydrolase Eij5 has preferable catalytic activity (Fig. 2) to the shorter lipid material of acyl group carbochain.

The analysis of 6 hydrolase Eij5 optimum reaction conditions of embodiment

Hydrolase Eij5 optimal reaction pH is measured in 3.0~10.5 ranges.Concrete operations are as follows: in different pH buffers 1mM p-nitrophenol butyrate and 4.04ng pure enzyme protein, the METHOD FOR CONTINUOUS DETERMINATION light absorption value A at 40 DEG C is added₃₄₈2min.Measurement makes Buffer are as follows: 100mM citric acid-sodium citrate buffer solution (pH 3.0~6.0), 100mM potassium dihydrogen phosphate-sodium hydroxide Buffer (pH 6.0~7.5), 100mM Tris hydrochloride buffer (pH 7.5~9.0) and 100mM 2- cyclohexylamino second sulphur Acid-sodium hydrate buffer solution (pH 9.0~10.5).Measurement result show Eij5 optimal reaction pH be 7.0, pH 6.5~ In 10.5 ranges active (Fig. 3).

Hydrolase Eij5 optimal reactive temperature measures within the scope of 15~60 DEG C.Concrete operations are as follows: in 1ml reaction system, 1mM p-nitrophenol butyrate, 100mM potassium dihydrogen phosphate-sodium hydrate buffer solution (pH 7.0) and the pure enzyme egg of 4.04ng is added It is white, METHOD FOR CONTINUOUS DETERMINATION light absorption value A under the conditions of 15,20,25,30,35,40,45,50,55 and 60 DEG C respectively₄₀₅2min.Measurement knot Fruit shows that the range of reaction temperature of Eij5 is 25~55 DEG C, and optimal reactive temperature is 40 DEG C (Fig. 4).

7 hydrolase Eij5 zymetology stability analysis of embodiment

The measurement concrete operations of bivalent cation and metal-chelator to hydrolase Eij5 activity influence are as follows: in reaction system In be separately added into 10mM Ba²⁺、Ca²⁺、Cd²⁺、Co²⁺、Cu²⁺、Mg²⁺、Mn²⁺、Ni²⁺、Sr²⁺、Zn²⁺And ethylenediamine tetra-acetic acid (EDTA), enzymatic activity is measured.Survey enzyme activity system are as follows: in 1ml reaction system, 1mM p-nitrophenol butyrate, 100mM phosphorus is added Acid dihydride potassium-sodium hydrate buffer solution (pH 7.0) and 4.04ng pure enzyme protein, the METHOD FOR CONTINUOUS DETERMINATION light absorption value A at 40 DEG C₄₀₅ 2min.Measurement result shows Eij5 activity in Ba²⁺、Ca²⁺、Mg²⁺And Sr²⁺In the presence of on enzyme activity influence less (retain 85% with Upper activity), in Mn²⁺、Zn²⁺、Co²⁺Stronger to its enzyme activity inhibiting effect under existence condition, Eij5 enzyme activity can be by Cd²⁺、Cu²⁺、Ni²⁺ Complete inhibition, EDTA can enhance its active (Fig. 5).

The measurement concrete operations of organic solvent and detergent to hydrolase Eij5 activity influence are as follows: distinguish in the reaction system 15% (v/v) organic solvent: acetone (Acetone), acetonitrile (Acetonitrile), ethyl alcohol (Ethanol), dimethyl methyl is added Amide (DMF), dimethyl sulfoxide (DMSO), glycerol (Glycerol), isopropanol (Isopropanol) and methanol (Methanol) or 1% detergent (w/v or v/v): SDS, polysorbas20 (Tween 20), Tween 80 (Tween80) and Triton The activity of X-100 measurement enzyme.Live body system are as follows: in 1ml reaction system, 1mM p-nitrophenol butyrate, 100mM phosphoric acid is added Potassium dihydrogen-sodium hydrate buffer solution (pH 7.0) and 4.04ng pure enzyme protein, the METHOD FOR CONTINUOUS DETERMINATION light absorption value A at 45 DEG C₄₀₅ 2min。 Measurement result show Eiij5 esterase can be kept under DMF, DMSO, glycerol, methanol existence condition 70% or more activity, SDS and Tween 20 can inhibit Eij5 esterase active (relative activity < 10%), and TritonX 100 can enhance its active (Fig. 6).

Sequence table

<110>the second institute of oceanography of Ministry of Natural Resources

<120>a kind of marine bacteria source development of new type alkali-resistant fibre organic solvent-resistant esterase and application

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 951

<212> DNA

<213> Erythrobacter jejuensis

<400> 1

atgaatgacg gcaatccctt cgtgcgcgat gatgtggcag ccttcctcgc cttgctggaa 60

caggcaggcg gcccgccgat caacgaagtc tcgctggagg aagcgcgcgg cgcctatatg 120

gcgctgcacc agatggctga ccgcccggcc cgtgatctcg ccgtgatcac cgatctgaca 180

tgcccaggcc cgggtggcga tatacccctg cgcctgtatg attcccgtga cagccgcgat 240

cctggcccgg tgatctgttt ctaccatggc ggcggattcg tgatcggcga tctcgatacc 300

caccacaatc tgtgcaccga aatcgcgcat cagatggatt tgcccgtggt ggcggtcgat 360

taccggctcg cgccggagca ccccttcccc gccccgatcg aggattgcat tgccgccagc 420

cgctggatcg ctgactcgcc cgaagcattg ggccgcccgg ccaccggcat tatcccgatt 480

ggcgatagcg cgggcggcaa tgcgaccatc gttgtaagcc aggcgctggc ccggcaaccg 540

gcggacacgc ctgtcctcct gcaggtgccg atcttcccgc tcgccagcga ctcggcaggc 600

tcaaccagcc tggaagaatt cgccgaggga tttgtcctca ccaaggtcgc gatcgaattc 660

ttcgaagcgg gctaccagcc cgacaaggcc gatccccgag ccatgccgat cctgggcagc 720

catgcgggca caccgccctc gatcgtagtc accgccagcc tcgatccgat ccgcgattcc 780

gggcgtgatt atgctgcggc actggcgcag gccgggattg atcatgtgtt cctggaggtt 840

gccggcggca ctcacagttt caccaatctg cgacaggcag tgcccagcta ccagaccgag 900

cttgagcggg tctttgccgc gatgaagctg atgctggcgg ggaacgcctg a 951

<210> 2

<211> 316

<212> PRT

<213> Erythrobacter jejuensis

<400> 2

Met Asn Asp Gly Asn Pro Phe Val Arg Asp Asp Val Ala Ala Phe Leu

1 5 10 15

Ala Leu Leu Glu Gln Ala Gly Gly Pro Pro Ile Asn Glu Val Ser Leu

20 25 30

Glu Glu Ala Arg Gly Ala Tyr Met Ala Leu His Gln Met Ala Asp Arg

35 40 45

Pro Ala Arg Asp Leu Ala Val Ile Thr Asp Leu Thr Cys Pro Gly Pro

50 55 60

Gly Gly Asp Ile Pro Leu Arg Leu Tyr Asp Ser Arg Asp Ser Arg Asp

65 70 75 80

Pro Gly Pro Val Ile Cys Phe Tyr His Gly Gly Gly Phe Val Ile Gly

85 90 95

Asp Leu Asp Thr His His Asn Leu Cys Thr Glu Ile Ala His Gln Met

100 105 110

Asp Leu Pro Val Val Ala Val Asp Tyr Arg Leu Ala Pro Glu His Pro

115 120 125

Phe Pro Ala Pro Ile Glu Asp Cys Ile Ala Ala Ser Arg Trp Ile Ala

130 135 140

Asp Ser Pro Glu Ala Leu Gly Arg Pro Ala Thr Gly Ile Ile Pro Ile

145 150 155 160

Gly Asp Ser Ala Gly Gly Asn Ala Thr Ile Val Val Ser Gln Ala Leu

165 170 175

Ala Arg Gln Pro Ala Asp Thr Pro Val Leu Leu Gln Val Pro Ile Phe

180 185 190

Pro Leu Ala Ser Asp Ser Ala Gly Ser Thr Ser Leu Glu Glu Phe Ala

195 200 205

Glu Gly Phe Val Leu Thr Lys Val Ala Ile Glu Phe Phe Glu Ala Gly

210 215 220

Tyr Gln Pro Asp Lys Ala Asp Pro Arg Ala Met Pro Ile Leu Gly Ser

225 230 235 240

His Ala Gly Thr Pro Pro Ser Ile Val Val Thr Ala Ser Leu Asp Pro

245 250 255

Ile Arg Asp Ser Gly Arg Asp Tyr Ala Ala Ala Leu Ala Gln Ala Gly

260 265 270

Ile Asp His Val Phe Leu Glu Val Ala Gly Gly Thr His Ser Phe Thr

275 280 285

Asn Leu Arg Gln Ala Val Pro Ser Tyr Gln Thr Glu Leu Glu Arg Val

290 295 300

Phe Ala Ala Met Lys Leu Met Leu Ala Gly Asn Ala

305 310 315

Claims (10)

1. a kind of isolated polypeptide with hydrolytic enzyme activities, is selected from the group:

(a) polypeptide, it is consistent with sequence shown in the polypeptide of SEQ ID NO:2；Or

(b) polypeptide is that the separate catalytic center position of polypeptide shown in SEQ ID NO:2 carries out various substitutions, addition and/or lacks The mutant that one or several amino acid obtain is lost, which has with protein sequence shown in SEQ ID NO:2 at least 90% or more homology and at least 90% or more hydrolytic enzyme activities.

2. polypeptide according to claim 1, it is characterised in that: the polypeptide with hydrolytic enzyme activities derives from bacterium Kind Erythrobacter jejuensis.

3. polypeptide according to claim 1, it is characterised in that: the catalytic center of the hydrolase is SEQ ID NO:2 institute 161-165,255 and No. 285 amino acid positions shown.

4. polypeptide according to claim 1, it is characterised in that: the mutant is polypeptide shown in SEQ ID NO:2 Various substitutions, additions and/or deletions are carried out less than the mutant that 5 amino acid obtain far from catalytic center position.

5. a kind of coding has the polynucleotides of polypeptide described in claim 1, it is selected from the group:

(a) polynucleotides, it is consistent with sequence shown in the nucleotide of SEQ ID NO:1；Or

(b) polynucleotides, for in nucleotide sequence shown in SEQ ID NO.1 remove 481-498,763-765 and 853-855 Other nucleotide outside the nucleotide of position are replaced, add and/or lack the mutant gene that one or several nucleotide obtain, The polynucleotides have the homology with nucleotide sequence at least 90% or more shown in SEQ ID NO:1.

6. a kind of nucleic acid construct, it includes the multicores for the claim 5 being operably connected with one or more regulating and controlling sequences Thuja acid, the regulating and controlling sequence instruct the generation of the polypeptide in suitable expressive host.

7. a kind of recombinant expression carrier, it includes the nucleic acid constructs of claim 6.

It is inverted by carrier as claimed in claim 7 or transfected prokaryotic is biological or eucaryote host obtains 8. a kind of host.

9. a kind of method for generating any one of the claim 1-4 polypeptide comprising:

(a), recombinant host cell according to any one of claims 8 is cultivated under conditions of helping to create hydrolase, wherein the place Chief cell includes nucleotide or the nucleotide in its at least one mutational site shown in SEQ ID N0:1；

(b), the polypeptide is recycled.

10. hydrolase described in claim 1 or the host according to any one of claims 8 that can express hydrolase are in catalysis ester-type hydrolysis In application.